1. Field of the Invention
The present invention relates generally to the field of molecular biology and functional genomics. More particularly, it concerns methods to screen genes or gene components for their functions or biological effects and methods of generating immune responses or immune reagents.
2. Description of Related Art
Genomic sequencing efforts are producing a wealth of data. Sequence information is being compiled not only from humans but also plants, animals and microbes. This abundance of data has spawned the need for new technologies for analyzing and functionally assessing the millions of genes that will now be available. The expanding repertoire of areas that functional genomics are applied should lead to new insights into evolution, reveal how cellular pathways integrate, and yield new drugs and vaccines. A current challenge is to develop the technologies that will enable these advancements.
For example, there are currently at least thirty microbial genome sequences in the public domain and additional projects underway. Most of these are pathogens of humans or commercial animals. One often expressed hope is that knowledge of these sequences will lead to the development of vaccines against these pathogens. Though computational analyses may be useful, the more sure-footed approach would be to functionally screen each gene from all the pathogens in animals for its value as a protective agent. However the time and expense of cloning thousands of genes from each pathogen then preparing appropriate reagents from them is prohibitive, using current methods.
In order to quickly and effectively assess the activity of any particular gene product or a physiological response to it, an assay method is required that avoids plasmids and bacterial cloning procedures. This ideal method would also permit the plethora of new genes from sequencing projects to be rapid screened in organisms, cells or cell-free systems.
The inventors have determined methods and compositions which allow for the production of linear expression elements (xe2x80x9cLEEsxe2x80x9d) and/or circular expression elements (xe2x80x9cCEEsxe2x80x9d) encompassing a complete gene (promoter, coding sequence, and terminator). These LEEs and CEEs can be directly introduced into and expressed in cells or an intact organism to yield expression levels comparable to those from a standard supercoiled, replicative plasmid.
In some general embodiments, the invention relates to methods of assaying for the production or regulation of expression of at least one polypeptide from a linear or circular nucleic acid segment comprising a promoter or putative promoter and an ORF or putative ORF. These methods may comprise: a) obtaining at least one linear or circular nucleic acid segment comprising a promoter or putative promoter and an ORF encoding a peptide or putative ORF; b) placing the nucleic acid segment under conditions conducive to expression of the polypeptide from the ORF; and c) assaying for the production or regulation of expression of a polypeptide from the ORF or putative ORF. In many preferred embodiments, the nucleic acid segment will comprise a terminator. However, in some applications, including those where the linear nucleic acid segment is assayed in a cell-free expression system, no terminator is required. In some cases, the linear nucleic acid is obtained by a PCR(copyright) process. In other situations, the linear nucleic acid may be cut out of a plasmid, chromosome, or other larger piece of nucleic acid; in these cases, the linear nucleic acid cut from the larger piece of nucleic acid will typically comprise a promoter and ORF, and in many cases a terminator, that are already in operable relationship.
Of course, those of skill in the art will understand that the promoter or putative promoter and any terminator will typically be placed in a operable relationship to the ORF or putative ORF, employing methods disclosed herein or know to those in the art.
In many cases, the linear nucleic acid segment is obtained by synthesis. In some preferred methods, the synthesis comprises non-covalent linkage of the promoter to the ORF. For example, this non-covalent linkage may be performed by a) obtaining a PCR(copyright) product comprising the nucleic acid segment, which PCR(copyright) product is obtained by amplification from at least one primer that has complementary stretches comprising deoxyuridines with uracil-DNA glycosylase to create overhangs to which the promoter can link; b) providing the promoter; and c) non-covalently linking the promoter to the nucleic acid segment to create the linear or circular expression element. In many cases a terminator will be non-covalently linked to the ORF, using a similar technique, although it is possible that the ORF or putative ORF may have a terminator incorporated into it, such that the addition of a terminator may not be required. In some presently employed embodiments, the primer that has complementary stretches comprising deoxyuridines. These stretches allow the use of uracil-DNA glycosylase, or another suitable enzyme to create overhangs to which the promoter can non-covalently link create the linear or circular expression element. The non-covalent linkage of a terminator to the ORF can be accomplished by much the same technique. Of course, it will be understood to those of skill in the art that other nucleotide/enzyme pairs may be used to perform this non-covalent linking, and that other techniques of non-covalent linking may be employed, so long as the purposes of the invention are accomplished. In some specific embodiments, the primer comprises the promoter and the terminator in divergent orientation, such that the step of non-covalently linking the promoter and the terminator to the ORF results in a circular expression element.
While the promoter may be of any origin that will work for the purposes of the invention, in some preferred embodiments, the promoter is a eukaryotic promoter. Likewise, the terminator may be of any source, but in may cases the terminator will be a eukaryotic terminator.
The nucleic acid segment containing the ORF, putative ORF, or any other nucleic acid segment which is comprised in a LEE or CEE may be obtained from any of a variety of sources. For example, it may be obtained by PCR(copyright), from a linear nucleic acid that is cut out of a plasmid, or obtained by synthesis.
In some methods according to the invention, the nucleic acid segment forming LEE or CEE is placed into a cell so that it is under conditions conducive to expression of a polypeptide from the ORF or putative ORF. In some preferred embodiments, the linear nucleic acid is placed into a cell but not integrated into the cell""s genome. The inventors have determined that integration into the genome is not required for expression of linear nucleic acids. Further, the inventors have determined that supercoiled plasmids are not required for expression of genes. In some embodiments, the cell is in cell culture, while in others, the cell is comprised in a tissue or an entire organism. All organisms are contemplated in this regard, including, but not limited to plant, animal, mammal, fish, bird, reptilian, human, rabbit, rat, hamster, mouse and other cells. The LEEs and CEEs of the invention may be placed into cells using any of the technologies described elsewhere in the specification. In some preferred embodiments, a LEE or CEE in injected into the cell. In some particularly preferred embodiments injection comprises microprojectile bombardment. In other embodiments, the LEE or CEE many be placed in a cell-free expression reaction.
Various preferred embodiments relate to methods of analyzing a nucleic acid sequence comprising: a) obtaining a nucleic acid segment; b) linking the nucleic acid segment to a promoter and a terminator to create a linear or circular expression element; c) providing the linear or circular expression element to a cell-free expression system or to a cell under conditions conducive to expression of any product encoded for by the nucleic acid segment; and d) analyzing any expression of any product encoded by the nucleic acid sequence. The nucleic acid segments employed in these methods can be obtained in manners described above, and elsewhere in the specification. Likewise the linkage of the promoter and terminator may be non-covalent linkage, as described elsewhere. In some embodiments wherein the nucleic acid sequence comprises an ORF to be analyzed for function, for example, to determine whether the nucleic acid encodes a polypeptide. These methods also allow one to determine whether or not the ORF encodes an antigenic polypeptide, and/or to determine biological properties of the polypeptide. For example one can determine whether the linear or circular expression element, or an ORF contained therein, is suitable for use in a vaccine. An advantage of these methods is that more than one distinct nucleic acid segment is analyzed in a single procedure.
As discussed above, and elsewhere in this specification, the methods of the invention may involve assaying for expression of a polypeptide which may be encoded in an ORF or putative ORF. In some preferred embodiments, the methods comprise assaying for expression of the polypeptide; in some cases, such assaying includes identification of the polypeptide. Other embodiments may comprise assaying the expression of a reporter gene product encoded in the ORF.
Still other specific embodiments comprise assaying for function of the promoter. For example, the function of the promoter may be assayed by determining whether a reporter gene product encoded in the ORF is expressed. A reporter gene is a gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional. Generally, reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture. In some embodiments, the assessment of promoter function will comprise comparing the function of two or more putative promoters for which function is assayed. In this manner, the invention provides for an efficient manner of screening a variety of promoters for function in a specific system. For example, a library of promoters from a variety of sources can be assayed such that an optimal promoter for a particular system is determined. Alternatively, a variety of mutants of a specific promoter may be assayed to determine whether they have promoter activity. In order to perform such assays, one may construct a variety of linear nucleic acid segments, using standard molecular biology means, each of which comprises a putative promoter or a promoter and a polypeptide encoding a reporter gene. This variety of linear nucleic acids may then be introduced to a system that enables the assessment of their function by looking for expression of the reporter gene.
In addition to allowing for analysis of ORFs or putative ORFs, the invention provides methods of analyzing a nucleic acid segment for activity as a promoter comprising: a) obtaining a nucleic acid segment encoding a putative promoter; b) linking the nucleic acid segment encoding the putative promoter to a nucleic acid encoding a polypeptide to create a linear or circular expression element; c) providing the linear or circular expression element to a cell-free expression system or to a cell under conditions conducive to expression of the polypeptide; and d) analyzing any expression of the polypeptide. For example, the function of the promoter may be assayed by determining whether a reporter gene product encoded in the ORF is expressed. A reporter gene is a gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional. Generally, reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture. In some embodiments, the assessment of promoter function will comprise comparing the function of two or more putative promoters for which function is assayed. In this manner, the invention provides for an efficient manner of screening a variety of promoters for function in a specific system. For example, a library of promoters from a variety of sources can be assayed such that an optimal promoter for a particular system is determined. Alternatively, a variety of mutants of a specific promoter may be assayed to determine whether they have promoter activity. In order to perform such assays, one may construct a variety of linear nucleic acid segments, using standard molecular biology means, each of which comprises a putative promoter or a promoter and a polypeptide encoding a reporter gene. This variety of linear nucleic acids may then be introduced to a system that enables the assessment of their function by looking for expression of the reporter gene. In this manner, one is able to assay for function of the promoter or putative promoter, and determine whether the promoter or putative promoter is functioning, and the extent of any such function, by determining whether the reporter gene product encoded in an ORF is expressed. In some embodiments, the methods may be used to compare the function of two or more putative promoters. It is advantageous that more than one distinct nucleic acid segment encoding a putative promoter can analyzed in a single procedure. The nucleic acid encoding the putative promoter may be, for example, a native nucleic acid, prepared by mutation of a native promoter sequence, or prepared by chemical synthesis. One of skill can use this these techniques to test and optimize promoters or putative promoters from any source.
In additional embodiments, the invention relates to methods of screening for a biological response comprising: a) obtaining a linear or circular expression element by a process comprising: obtaining a DNA segment comprising an ORF; linking the ORF to a promoter and a terminator to create a linear or circular expression element; and b)providing the expression element to an organism under conditions conducive to expression of any product encoded for by the ORF. In such cases, more than one type of linear or circular expression element is introduced to the organism. These methods may encompass method of producing antibodies for analytical purposes, for example, when the biological function is to result in an immune response. In other embodiments, these methods may provide methods of vaccinating the organism, as described below.
In some specific embodiments, the invention allows for the vaccinating an organism comprising obtaining a linear or circular expression element by a process comprising obtaining a DNA segment comprising an ORF and linking the ORF to a promoter and a terminator to create a linear or circular expression element; and c) providing the expression element to an organism under conditions conducive to expression of any product encoded for by the ORF, such that immune response is produced in the animal. In such vaccines, more than one type of linear or circular expression element is introduced to the organism, in order to create a polyvalent vaccine that my be directed against more that one infectious agent, cancer, or other disease or against a variety of antigens from a single infectious agent, cancer, or other disease. For example, a plurality of types of linear or circular expression elements may introduced to the organism., and the plurality of types of linear or circular expression elements may comprise elements encoding distinct polypeptides of a pathogen. While those of skill in the art will realize that the pathogen may be of any form, in some preferred embodiments, the pathogen is a virus, bacterium, fungus, alga, protozoan, arthropod, nematode, platyhelminthe, or plant. In even more specific embodiments, the individual linear or circular expression elements encoding all potential allergens of a virus is comprised in the plurality of types of linear or circular expression elements. The ORF may also encodes a polypeptide which is useful for vaccination against cancer, or another disease, as known to those of skill in the art.
The invention also contemplates methods of selecting ORFs effective for generating an immune response specific to a pathogen, cancer, or other disease in an organism, comprising: a) preparing a plurality of linear or circular expression elements comprising a plurality of DNA segments comprising ORFs from a pathogen, cancer, or other disease; b) introducing the plurality of linear or circular expression elements into an organism; and c) selecting from the plurality of linear or circular expression elements ORFs that are effective to generate said immune response. Such methods may further comprising testing said organism against challenge with the pathogen wherein the organism is protected against challenge with the pathogen. In this manner, one or more antigens conferring a protective response may identified by screening of the organism.
The invention also relates to linear and circular expression element and method of producing linear and circular expression elements. For example, such methods may comprise a) obtaining a DNA segment comprising an ORF, putative ORF, or other sequence; and b) linking the DNA segment to a promoter and a terminator to create a linear or circular expression element. In some cases, the DNA segment is obtained from a process involving PCR(copyright), and in some specific embodiments, the PCR(copyright) reaction is primed with oligonucleotides having a complementary stretch incorporating deoxyuridines. For example, the deoxyuridines may be incorporated every third position of the complementary stretch. The ORF may be non-covalently linked to the promoter and the terminator, and this non-covalent linkage may be performed as described elsewhere in this specification.
In some specific embodiments, the invention relates to linear or circular expression elements comprising a DNA segment comprising an ORF and a promoter and terminator non-covalently linked to said ORF. In other aspects, the invention relates to antibodies, antigens and vaccines that are prepared, assayed, or determined employing the above-described methods.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used herein in the claim(s), when used in conjunction with the word comprising, the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one.